The opioid epidemic and prevalence of chronic pain in the United States have generated a critical need for new pain therapeutics with limited side effects. Pain descending control systems, which consist of brainstem neural circuits projecting to the spinal cord, critically modulate nociception in the spinal dorsal horn and may contribute to chronic pain. Previous studies established that these descending circuits include several populations of rostral ventromedial medulla (RVM) neurons. RVM neurons were originally classified based on their electrophysiological properties. Specifically, ON-cells exhibit increased activity in response to noxious stimuli and facilitate spinal pain transmission. In contrast, RVM OFF-cells display high basal tonic firing that is suppressed following noxious stimulation and exert inhibitory pain control. Despite their importance, RVM nociceptive neurons remain understudied and poorly defined at the molecular level compared to nociceptors and spinal neurons, preventing the targeting of descending control systems for pain management. This research proposes to combine neural circuit tracing, mouse genetics, and single cell RNA-sequencing (scRNA-seq) to identify marker genes that define nociceptive RVM neuronal types for functional studies. Aim 1 will determine the molecular signatures of spinally projecting RVM?SC ON-cells. To profile RVM?SC neurons, we inject a retrograde reporter virus (AAV- retro-CAG-GFP) into the dorsal horn, and use fluorescence-activated cell sorting (FACS) to isolate GFP+ retro- labelled RVM?SC neurons from RVM and perform scRNA-seq. Unbiased clustering of cells will identify numerous RVM neuronal cell types classified by expression of specific marker genes. Aim 2 will identify the RVM neurons that are active during pain. The expression of the immediate early gene c-fos has been used extensively for the mapping of CNS nociceptive neurons. To label RVM neurons active during pain, we use the recently developed FosTRAP2 mice, in which the promoter of c-fos drives the expression of CreERT2 recombinase, in combination with fluorescent Cre-reporter mouse lines or viruses. FACS will isolate fluorescently labeled RVM neurons, and scRNA-seq will identify the neuronal types activated during nociceptive stimulation. Finally, the RVM undergoes functional reorganization during chronic pain or opioid exposure. Aim 3 will thus determine the transcriptional changes in RVM?SC neurons associated with the transition from acute to chronic pain, and the emergence of side effects during chronic opioid treatment. We will use approaches described in Aim 1 in combination with chronic pain and opioid exposure models to resolve the transcriptional changes that occur in RVM?SC neurons during the development of these pathological hyperalgesic states. This research will considerably broaden our understanding of pain neurobiology, by providing a categorization of new markers that define classes of RVM neurons for the future functional identification of the discrete brainstem circuits that influence pain perception and opioids effects. Furthermore, this research may identify novel molecular targets in RVM neurons for the development of innovative analgesic strategies that modulate activity in descending pain control systems.